Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum

Summary The distribution of spontaneous sister chromatid exchanges (SCEs) was studied in PHA-stimulated lymphocytes from 15 patients affected by xeroderma pigmentosum (XP). The study of unscheduled DNA synthesis (UDS) in twelve of these patients showed that seven were deficient and five proficient. The number of SCEs in XP patient cells was higher than in those of 19 controls, and the distributions of SCEs per cell were significantly different. However, the results varied when XP patients were considered in relation to their UDS: the group of XP patients with proficient UDS did not differ, whereas the group of XP patients with deficient UDS was very significantly different from controls. The group not tested for UDS was similar to the deficient UDS group. The possible relationship between the increase of SCEs and the type of DNA repair defect is discussed.     

Induction of sister chromatid exchanges in xeroderma pigmentosum cells after exposure to ultraviolet light

The role of DNA repair mechanisms in the induction of sister chromatid exchanges (SCE) after exposure to ultraviolet radiation was investigated in xeroderma pigmentosum cells. Cells from different excision-deficient XP strains, representing the 5 complementation groups in XP, A, B, C, D and E, and from excision-proficient XP variant strains were irradiated with low doses of UVR (0-3.5 J/m2). The number of SCE was counted after two cycles in the presence of BUdR. In cells of the complementation groups A, B, C and D the number of SCE was significantly higher than in UV-exposed control cells. The frequencies of SCE in group E cells and in XP varient cells were not different from those in control cells. Treatment with caffeine (0-200 microgram/ml) did not result in a different response of variant cells compared with normal cells. A simple correlation between SCE frequency and residual excision-repair activity was not observed. The response of the excision-repair deficient cells suggest that unrepaired damage, produced by UVR is involved in the production of SCE.     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

Non-uniform distribution of sister chromatid exchanges in human lymphocytes

To test whether sister chromatid exchange (SCE) scores on human chromosomes have a uniform distribution, simulated SCE scores were generated and compared with observed scores using log-linear models. The analysis was performed at the level of the chromosome groups. Using this method we first tested whether the number of SCEs was distributed uniformly, i.e. proportional to the relative length of the chromosomes. Refinements of this hypothesis were made by considering a variable region around a first SCE to be inert for other SCEs and by making the occurrence of an SCE on a chromosome dependent on the occurrence of another SCE on the same chromosome. In further analyses it was tested whether the number of SCEs was proportional to the number of G bands on a chromosome, or to the DNA content of the chromosomes. None of the tested hypotheses fitted the observed data, establishing the non-uniform distribution of these events.     

Ultrasonic induction of sister chromatid exchanges in human lymphocytes

Summary We analyzed sister chromatid exchange (SCE) frequencies as an indicator of DNA damage induced in human lymphocytes by real-time ultrasound. A range of exposure times and intensities was tested in a series of blind, randomized, in vitro experiments under spatial and sonographic conditions simulating exposure of a gravid abdomen and uterus. Our studies showed small but consistent effects of ultrasound on SCE frequencies, for each experiment. Differences between matched control and exposed means were significantly different from zero. X ² tests for homogeneity indicated no significant differences among either the means or the total distributions of the controls, nor among each of the separate dose levels. Consequently, experiments were pooled, and X ² analysis indicated significant differences both among distributions and among means of SCE frequencies for controls versus exposed cells (P(0.001). The pooled control mean was also significantly different from each of the pooled dose means. Correcting for multiple comparisons gave identical results for the paired comparisons of means except for the 20-min level which was borderline (0.025P(0.01). We conclude that the well-established value of clinical ultrasonography warrants its continued use; however, minimizing the numbers and lengths of exposure per patient would seem prudent, pending further information on clinical implications of our results.Supported in part by NIH-HD82855 and HD 11021 and a National Foundation Summer Science Research Grant for Medical Students, 8-80-22     

A schedule to demonstrate radiation-induced sister chromatid exchanges in human lymphocytes

Summary The reciprocal interchange between the chromatids of a chromosome, termed sister chromatid exchange (SCE), is considered to be one of the most sensitive and accurate cytogenetic parameters and respond to toxic chemicals at very low doses. But the response of SCE to ionizing radiation is very poor. Human lymphocytes fail to give SCE response when irradiated at G₀. Probably the primary lesions induced at G₀ do not remain available long enough to find expression as SCEs. Based on this assumption a schedule was developed using caffeine to demonstrate radiation induced SCEs. Following this schedule a dose-dependent increase in the frequency of radiation induced SCEs has been observed.     

Benzpyrene-Induced sister chromatid exchanges in lymphocytes of patients with lung cancer

Summary The effect of benzpyrene on sister chromatid exchange was determined in PHA-stimulated lymphocytes of 18 patients with lung cancer and 11 controls without cancer or bronchopulmonary diseases. Patients and controls did not differ either with respect to the spontaneous rate of sister chromatid exchanges or in their response to the carcinogen. We conclude that individual susceptibility to lung cancer cannot be detected by an individual response to benzpyrene, at least in lymphocytes and at the chromosomal level.     

Dose dependence of sister chromatid exchanges in human lymphocytes induced by in vitro α-particle irradiation

Exposure of human G₀ lymphocytes to high-LET particles under different conditions has been seen, unlike low-LET radiations, to be substantially effective in the induction of sister chromatid exchanges (SCE). However, whereas for fast neutrons a linear dose response of SCE has been determined, there is no sign of a dose-response relationship for α-particles. A likely reason for this lack of dose dependence may be the irradiation procedure. Therefore, a technique developed in our laboratory to ensure uniformity of irradiation with α-particles was used in the present study. Monolayers of 3 h-stimulated lymphocytes were exposed with α-particles from ²⁴¹Am. Underdispersion was found for the cell-to-cell variance of the number of SCE. The dose response of SCE was linear, with a yield of 3.4 SCE per cell and per Gray. Received: 26 April 1996 / Accepted in revised form: 24 July 1996     

Propofol anesthesia in children does not induce sister chromatid exchanges in lymphocytes

Background: Propofol is frequently used for general anesthesia in children although little is known about possible genotoxic effects in humans. We investigated the formation of sister chromatid exchanges (SCE) in metaphase chromosomes of T-lymphocytes of children as a marker for possible genotoxocity following total intravenous anesthesia with propofol for minor surgical procedures.Methods: 40 children ASA classification I–III were included (ASA I n=34, ASA II n=5, ASA III n=1) in the study. Anesthesia was induced by propofol (3 mg/kg) and alfentanil. Succinylcholine or rocuronium were administered for muscle relaxation. After tracheal intubation anesthesia was maintained by continuos propofol infusion at 12 mg/(kg h). Blood samples were drawn before induction and after termination of anesthesia. Following a 72 h cell culture period, 25 T-lymphocyte metaphases per blood sample for all children were analyzed for SCE frequencies.Results: Total intravenous anesthesia with propofol on children did not influence SCE rates in metaphase chromosomes of T-lymphocytes. No SCE differences could be detected between blood samples before initiation and after termination of anesthesia (Wilcoxon signed rank test). Slightly elevated SCE rates were obtained in T-lymphocytes of girls compared to boys, but these differences did not reach statistical significance.Conclusions: Propofol anesthesia under the chosen conditions did not induce the formation of SCE in children in vivo. No genotoxic effect of a short term exposure to propofol during pediatric anesthesia had been observed.     

Propofol anesthesia in children does not induce sister chromatid exchanges in lymphocytes

BACKGROUND: Propofol is frequently used for general anesthesia in children although little is known about possible genotoxic effects in humans. We investigated the formation of sister chromatid exchanges (SCE) in metaphase chromosomes of T-lymphocytes of children as a marker for possible genotoxocity following total intravenous anesthesia with propofol for minor surgical procedures. METHODS: 40 children ASA classification I-III were included (ASA I n=34, ASA II n=5, ASA III n=1) in the study. Anesthesia was induced by propofol (3mg/kg) and alfentanil. Succinylcholine or rocuronium were administered for muscle relaxation. After tracheal intubation anesthesia was maintained by continuous propofol infusion at 12 mg/(kgh). Blood samples were drawn before induction and after termination of anesthesia. Following a 72 h cell culture period, 25 T-lymphocyte metaphases per blood sample for all children were analyzed for SCE frequencies. RESULTS: Total intravenous anesthesia with propofol on children did not influence SCE rates in metaphase chromosomes of T-lymphocytes. No SCE differences could be detected between blood samples before initiation and after termination of anesthesia (Wilcoxon signed rank test). Slightly elevated SCE rates were obtained in T-lymphocytes of girls compared to boys, but these differences did not reach statistical significance. CONCLUSIONS: Propofol anesthesia under the chosen conditions did not induce the formation of SCE in children in vivo. No genotoxic effect of a short term exposure to propofol during pediatric anesthesia had been observed.     

5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes

Summary When [³H]dC was added with a high dose (4x10^-1 mM) of dT to human blood lymphocyte cultures, much heavier labeling of interphase nuclei and metaphase chromosomes was observed compared with that in cultures treated with [³H]dC alone. This observation indicates that in the presence of excess dT, exogenous dC is included into cytosine bases of DNA, releasing the cells from the thymidine block.BrdC 5x10^-2 mM added with a high dose of dT (4x10^-1 to 1.0 mM) to the cultures did not relieve the thymidine block as determined from the percentage of metaphases of the first to third divisions. It is concluded that BrdC, in contrast to dC, is not utilized as a cytosine DNA precursor even in the presence of high concentrations of dT.The frequency of SCEs per cell was the same when studied with the aid of BrdC and BrdU used under similar conditions. The distribution of SCEs among chromosomes was also identical for both analogues: The number of SCEs was significantly higher than expected in chromosomes of group B and lower than expected in chromosomes of groups E, F, and G.     

Spontaneous and induced sister chromatid exchanges in the blood lymphocytes of healthy persons and of xeroderma pigmentosum patients exposed to the inhibitors of DNA repair and replication caffeine, 3-methoxybenzamide and novobiocin

The frequency of sister chromatid exchanges (SCEs), both spontaneous and induced by UV-light, X-rays, mitomycin C and ethylmetansulphonate (EMS), has been investigated in cultured human peripheral blood lymphocytes. Besides, frequency of spontaneous and induced SCEs was studied under the action of the inhibitors of topoisomerase II, polymerase poly(ADP-ribose), and DNA repair, i. e. novobiocin, 3-metoxybenzamide, and caffeine, respectively. It is shown that the base-line SCEs in lymphocytes of the patient with xeroderma pigmentosum II (XP2LE) is dramatically higher compared to that in normal and pigmented xerodermoid cells (XP3LE). The above inhibitors of DNA synthesis and repair enhance the rate of spontaneous SCEs in normal, XP2LE and XP3LE cells. UV-, X-ray and chemical mutagens induced an increased frequency of SCEs in these cells. Simultaneous treatment with mutagenes and inhibitors of DNA synthesis and DNA repair enhanced the rate of SCEs in lymphocytes of healthy donors and in the XP3LE patient. The frequency of the XP2LE cells. Novobiocin, 3-MBA and caffeine significantly decreased the frequency of SCEs in mitomycin C- and EMS-treated XP2LE lymphocyte, which nevertheless was much higher than that in normal cells treated with the same agents.     

Analysis of sister chromatid exchanges in lymphocytes cultured from 71 healthy men

Sister chromatid exchanges (SCE) were analyzed in peripheral blood lymphocytes from a select group of 71 healthy men, 56 nonsmokers and 15 cigarette smokers. In addition to estimating baseline SCE, data were examined to seek relationships of SCE frequencies to age and smoking. The baseline value of 7.53 SCE per cell from the 56 nonsmokers was within the range (5.60 to 9.10 SCE/cell) reported for other human populations. No relationship was found between the mean SCE frequency per cell and age. However, a significant increase in the SCE mean value was observed in smokers as compared to nonsmokers. The results of this study are compared with those of other reports on SCE effects of age and smoking.Abbreviations BUdR 5-bromo,2-deoxyuridine - SCE sister chromatid exchange     

Reduced frequencies of Mitomycin-C induced sister chromatid exchanges in AKR Mice

Summary The frequencies of base-line and Mitomycin-C (MMC) induced sister chromatid exchanges (SCE) were surveyed in four inbred strains of mice. In contrast to the C57B1/6J, CBA/J, and A/J strains where frequencies of SCE increased linearly with increasing dose of MMC, levels of SCE were significantly lower in AKR/J mice at high MMC concentrations. At a dose of 5 mg/kg MMC, chromosomal aberrations were more frequent in bone marrow cells of AKR/J mice than in C57B1/6J mice. These observations suggest an altered response to DNA damage in the AKR mouse strain.     

Statistical evaluation of sister chromatid exchanges

Summary Log-linear models are fitted to sister chromatid exchange (SCE) scores in order to test the significance of the differences in SCE scores observed between individuals or between experimental treatments. The analysis is performed at the level of chromosome groups. In each single test all measurements from all chromosome groups, both from the control and from the experimental sets, are utilized. By proceeding in this way full use is made of all the available information on the SCE scores at the level of chromosome groups and the shortcomings of the classical Student-t and chi-square tests are avoided.This work was supported by a grant Geconcerteerde Acties from the Belgian Government.     

Frequency of sister chromatid exchanges in human lymphocytes at different times of day

Analysis of the frequency of sister chromatid exchanges in the human blood taken from normal female donors was made at 9 a. m. and 9 p. m. The morning rate of exchange is shown to be less than the evening one. The influence of physiological state of the organism on the sister chromatid exchange is suggested.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

Adaptation-like response to the chemical induction of sister chromatid exchanges in human lymphocytes

Summary Experiments have been performed to determine whether human lymphocytes in primary cultures can show an adaptive response to the induction of cellular lesions (manifested as a production of sister chromatid exchanges, SCEs) as previously found in bacteria and established human and mammalian cell lines. Human lymphocytes were pretreated with various subtoxic concentrations (5–50ng/ml) of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) once every 6h for 72h, and subsequently challenged by a high dose (4g/ml) of MNNG. The lymphocytes in MNNG-challenged cultures had the lowest frequency of SCEs when pretreated with 10ng/ml MNNG. Further cross-resistance study revealed that repeaied pretreatments of lymphocytes with 10ng/ml MNNG for 72h can render the cells resistant to the induction of SCEs by the following challenge with a high dose of MNNG, but not of mitomycin C or ethyl nitrosourea. The data also suggest variations in the degree of the adaptation-like response among individuals.